PGlo Transformation

Purpose

The purpose of this experiment was to test the idea that cells can take in genetic information. By using the glo gene, and putting these cells in different situations we were able to see what it takes for a cell to incorporate foreign DNA into their own DNA. This also helped us learn how viruses are able to take over an organism.  

Introduction 

In this experiment we did genetic transformation. Genes contain pieces of DNA that provides the information to make the proteins. Proteins gives an organism their trait. When changed caused by genes it is consider genetic transformation and many use in science. One application would be biotechnology in the fields of agriculture and medicine. For example, with bacteria by moving genes with the help of the plasmid, which the circular DNA. Plasmid DNA usually contains more than one trait that helps the survival of bacteria. Bacteria can transfer plasmids to adapt to new environment. Bacteria becomes resistance to antibiotics because of this transfer. Green fluorescent protein or GFP can by transformed by plasmids. It causes them to glow. When the transformation is done the bacteria will glow fluorescent in the dark. The PGLO plasmid codes for this gene that is resistant to antibiotic ampicillin. To control the rate of proteins that transferred into a cell caused by gene regulation system. Sugar arabinose can be active by GFP. This procedure takes place on antibiotic growth plate

Methods

First, we put in transformation liquid into
each tube; one labeled +pGlo and another
-pGlo.

Then we put them on ice .

Next, we transfered e coli to the tubes. 

We added plasmid DNA only to the
+pGlo tube.

Then we cold shocked the tubes for
10 minutes.

While we waited we labeled our petri dishes

Next we heat shocked the cells by putting
the tubes in a water bath, and back again into
the ice.

Then we added LB nutrient broth to the
mixtures, and mixed.

Lastly, we spreaded the solutions onto
the petri dishes, and let them
incubate for one day. 

Data and Graphs:

Discussion:

The LB/-PGLO plate had the most bacteria that resembled the original untransformed E. coli colonies we initially observed. This makes sense, if we take into consideration the fact that the bacteria were removed from the starter plate, did not have any plasmid added to them, and only had LB (this is broth, or in simpler terms, food) on the plate. Regulating these features makes it a control plate. The other control plate is the LB/amp/-PGLO plate, which had zero growth, because no plasmid was added. Plasmid actually has to be added in order for bacteria to multiply in the presence of ampicillin. The transformation plates include LB/amp/+PGLO and LB/amp/ara/+PGLO. First, both plates had the plasmid for the fluorescent gene added to the E. Coli spread on them. Because of the heat shock we gave the E. Coli, holes could be made in the cell membrane of the prokaryote cells. This means that the PGLO plasmid which expresses the ampicillin resistance gene is incorporated into the E. Coli. Thus the bacteria can survive on the plates that contain ampicillin. On the other hand, cells that were not treated with DNA did not express ampicillin resistance and did not grow on the LB/amp/-PGLO plate. The LB/amp/ara/+PGLO plate had about the same amount of growth as the other transformation plate, but the genes of the plasmid will only be expressed in the presence of arabinose, so it’s on this plate that the bacteria glowed under a UV light. The transformation efficiency (efficiency by which cells take in extracellular DNA and express its genes) for this plate is 406.25 transformants/microgram. This basically reflects how competent prokaryotic cells are at including new DNA. Our results are what they were supposed to be, and they support our belief that only the cells with DNA added to them would be genetically transformed. One way to improve this lab could be to let it go on for more than one day. That way we would try to calculate rate of reproduction in order to grasp how quickly plasmids can be generated in new cells.

Conclusion :

 We found that we were able to transform the DNA of this organism using plasmids. We concluded that E. Coli did not grow. Some bacteria was able to live in the surroundings with ampicillin  and glow under UV light in a environment with arabinose. Based on these  result our  hypothesis came true.